Diagnostic biomarker to predict women at risk for preterm delivery

ABSTRACT

The invention relates to biomarkers associated with preterm delivery. More specifically, the invention provides methods of measuring biomarkers including but not limited to cytokines, cytokine receptors, cytokine receptor antagonists, chemokines, chemokine receptors, and/or chemokine receptor antagonists found in women that are at risk for preterm delivery. The diagnostic methods may be performed on whole blood.

FIELD OF INVENTION

This invention relates to preterm delivery. More specifically, theinvention provides biomarkers and methods of using biomarkers fordetermining preterm delivery risk.

BACKGROUND

All publications herein are incorporated by reference to the same extentas if each individual publication or patent application was specificallyand individually indicated to be incorporated by reference. Thefollowing description includes information that may be useful inunderstanding the present invention. It is not an admission that any ofthe information provided herein is prior art or relevant to thepresently claimed invention, or that any publication specifically orimplicitly referenced is prior art.

Preterm delivery is one of the most important fetal health problems inthe United States today. Approximately one in eight newborns isdelivered preterm and the incidence of prematurity has not decreased inthe last 20 years. Most preterm babies, if they survive, often havecardiac, neurologic, ophthalmic, and gastrointestinal problems that canextend even beyond childhood, and perhaps lead to adult diseases such asatherosclerosis. Currently, there are few, if any diagnostic biomarkersavailable that effectively identify women who are going to deliverpreterm. Biomarkers that are able to identify these women at risk wouldbe useful in the deployment of prevention/treatment strategies and toassess the effectiveness of these treatments. Thus, there is a need todevelop novel diagnostics that may identify women who will deliverpreterm. Especially, diagnostic biomarkers that may be detected innon-pregnant women or in women during the first trimester. Thebiomarkers may also be used later in the pregnancy to assess theeffectiveness of treatment and/or prevention strategies.

SUMMARY OF THE INVENTION

The following embodiments and aspects thereof are described andillustrated in conjunction with compositions and methods which are meantto be exemplary and illustrative, not limiting in scope.

Various embodiments of the present invention provides for a method,comprising: obtaining a biological sample from a woman who desires anassessment of her risk for preterm delivery; exposing the biologicalsample ex vivo to an antigen and/or a stimulant; and assessing an immuneresponse to predict whether the woman is at risk for preterm delivery.In various embodiments, the biological sample can be whole blood.

In various embodiments, the method can further comprise separating bloodcells from the sample and exposing the separated blood cells to theantigen.

In various embodiments, exposing the biological sample ex vivo to theantigen or the stimulant can be by collecting the biological sample intoa container coated with the antigen and/or stimulant.

In various embodiments, the method can further comprise removing plasmafrom the biological sample and assaying the plasma to assess the immuneresponse. In various embodiments, removing plasma from the biologicalsample can comprise centrifuging the tube comprising the biologicalsample and harvesting the plasma.

In various embodiments, the antigen and/or stimulant can be cortisol,LPS, phytohemagglutinin, a maternal histocompatibility antigen, apaternal histocompatibility antigen, a fetal histocompatibility antigen,a microbial antigen, a cytokine, a phorbol ester, an agent capable ofinducing Th1 or Th2 type differentiation of lymphocytes or combinationthereof.

In various embodiments, assessing the immune response can compriseassaying an expression level of a cytokine, cytokine receptor, cytokinereceptor antagonist, chemokine, chemokine receptor, chemokine receptorantagonist, or combinations thereof. In various embodiments, thecytokine, cytokine receptor, cytokine receptor antagonist, chemokine,chemokine receptor, chemokine receptor antagonist, or combinationsthereof can be selected from the group consisting of IL-10, IL-13,IL-1RA and combinations thereof.

In various embodiments, a higher or lower expression level of thecytokine, cytokine receptor, cytokine receptor antagonist, chemokine,chemokine receptor, and/or chemokine receptor antagonist compared to acontrol can indicate that the woman is at an increased risk of pretermdelivery. In various embodiments, the higher or lower expression levelof the cytokine, cytokine receptor, cytokine receptor antagonist,chemokine, chemokine receptor, and/or chemokine receptor antagonist canbe at least one to three times higher or lower compared to the levels ofthe control. In various embodiments, the level of the higher or lowerexpression of the cytokine, cytokine receptor, cytokine receptorantagonist, chemokine, chemokine receptor, and/or chemokine receptorantagonist can be indicative of a low, medium, or high risk of pretermdelivery. In various embodiments, a statistically significantly higheror lower expression of the cytokine, cytokine receptor, cytokinereceptor antagonist, chemokine, chemokine receptor, and/or chemokinereceptor antagonist compared to a control can indicate that the woman isat an increased risk of preterm delivery.

In various embodiments, assessing the immune response can comprise usinga technique selected from the group consisting of enzyme-linkedimmunosorbent spot (ELISpot), enzyme-linked immunosorbent assay (ELISA),microarray assay, quantitative BCP, Northern blot assay, Southern blotassay, Western blot assay, immunohistochemical assay, binding assay andcombinations thereof.

Various embodiments provide for a kit, comprising: a containercomprising an antigen and/or a stimulant; instructions to use thecontainer to collect a biological sample from a woman to detect animmune response; and instructions to determine the risk of pretermdelivery of the woman. In various embodiments, the kit can furthercomprise a reagent to detect the immune response. In variousembodiments, the kit can further comprise a positive control and/or anegative control.

Various embodiments provide for a method, comprising: obtaining a sampleof whole blood from a woman who desires an assessment of her risk forpreterm birth; exposing the sample ex vivo to an antigen and/or astimulant by collecting the sample into a container coated with theantigen and/or stimulant; and assessing an immune response to predictwhether the woman is at risk for preterm birth. In various embodiments,the antigen and/or stimulant can be cortisol, LPS, phytohemagglutinin,or combinations thereof. In various embodiments, assessing the immuneresponse can comprise assaying an expression level of a cytokine,cytokine receptor, cytokine receptor antagonist, chemokine, chemokinereceptor, chemokine receptor antagonist, or combinations thereof,wherein a statistically significantly higher or lower expression levelof the cytokine, cytokine receptor, cytokine receptor antagonist,chemokine, chemokine receptor, and/or chemokine receptor antagonistcompared to a control indicates that the woman is at an increased riskof preterm delivery. In various embodiments, the cytokine, cytokinereceptor, cytokine receptor antagonist, chemokine, chemokine receptor,chemokine receptor antagonist, or combinations thereof can be selectedfrom the group consisting of IL-10, IL-13, IL-1RA and combinationsthereof.

Other features and advantages of the invention will become apparent fromthe following detailed description, taken in conjunction with theaccompanying drawings, which illustrate, by way of example, variousfeatures of embodiments of the invention.

BRIEF DESCRIPTION OF THE FIGURES

Exemplary embodiments are illustrated in referenced figures. It isintended that the embodiments and figures disclosed herein are to beconsidered illustrative rather than restrictive.

FIGS. 1A and 1B depict graphical representation of expression levels ofvarious biomarkers of preterm individuals as compared to full termindividuals in accordance with various embodiments of the presentinvention. Empirical means and standard errors for each cytokine andtreatment group are presented.

DESCRIPTION OF THE INVENTION

All references cited herein are incorporated by reference in theirentirety as though fully set forth. Unless defined otherwise, technicaland scientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs. One skilled in the art will recognize many methods andmaterials similar or equivalent to those described herein, which couldbe used in the practice of the present invention. Indeed, the presentinvention is in no way limited to the methods and materials described.

Multiple factors lead to preterm delivery; however, immune activation isthought be the final effector pathway that leads to preterm rupture ofmembranes and contractions. Family and genetic studies suggest thatprematurity runs in families and history of prior preterm deliveryincreases the risk of future preterm deliveries. The inventors believedthat these observations can be explained by the inherent differences inthe immune responses of women who deliver preterm compared with thosewho deliver full term, and that these differences can be detected evenin the non-pregnant state. The inventors believed that by detectingthese differences one may be able to identify those women who are atrisk to deliver preterm even before they become pregnant. The inventorsbelieved that in order to see the differences, the immune cells of thepatient had to be stressed.

The inventors are the first to have discovered a biomarker assay thatprovides physicians with a tool to identify women who are at risk forpreterm delivery, even before they become pregnant or while pregnant,using a nonspecific immunologic test. Identification of women at riskallows the physician to better focus on preventative strategies in thesewomen and improve pregnancy outcome. In one embodiment, the test may bedone on small amounts of blood sample obtained from the subject, thusbeing minimally invasive to the fetus. The blood may be drawn directlyinto a test tube coated with a stimulant; alternatively the blood may besent to a laboratory to be processed and stimulated in the laboratory.Furthermore, the test may be repeated multiple times during the courseof pregnancy. Thus, it provides dynamic assessment of the pretermdelivery risk under the influence of changing environmental/physiologicfactors, as wells as requires minimal skills to draw blood as opposed toobtaining amniotic fluid.

One skilled in the art will recognize many methods and materials similaror equivalent to those described herein, which could be used in thepractice of the present invention. The present invention is in no waylimited to the methods and materials described. For purposes of thepresent invention, the following terms are defined below.

As used herein, the term “preterm delivery” refers to a premature birthor conditions associated with a premature birth, including for example,a child delivered before 34 weeks of gestation.

“Biomarker,” “diagnostic biomarker,” or “preterm delivery biomarker”refers to a molecular indicator that is associated with a particularpathological or physiological state. The “biomarker” as used herein isan indicator for risk of preterm delivery. The indicator can be acytokine, cytokine receptor, cytokine receptor antagonist, chemokine,chemokine receptor, chemokine receptor antagonist or an immunomodulatingagent, including interleukins and interferons. Examples of “biomarkers”include but are not limited to IL-2, IL-4, IL-5, IL-8, IL-10, IL-12,IL-13, GM-CSF, IFN-g, and TNF-a. Preferably, biomarkers of the presentinvention include IL-10, IL-13 and/or IL-IRA. A “biomarker” of thepresent invention may be detected in a sample.

“Sample” or “a biological sample” refers to cells or component parts, ora fraction or portion thereof of body fluids, including but not limitedto blood (e.g., whole blood), amniotic fluid, or cord blood. A “sample”or “biological sample” further refers to plasma, serum, and/orperipheral blood mononuclear cells (PMBC).

In one embodiment, the present invention provides a method of diagnosingsusceptibility for preterm delivery in non-pregnant women or pregnantwomen comprising obtaining a sample from the woman and assaying thesample for the presence or absence of one or more diagnostic biomarkers,where the presence or absence of one or more diagnostic biomarkers isindicative of susceptibility for preterm delivery in the woman. Inanother embodiment, the sample comprises PMBC supernatant. In anotherembodiment, the sample comprises whole blood. In another embodiment, thesample comprises serum. In another embodiment, the one or morediagnostic biomarkers comprise a microbial component lipopolysacharide(LPS) or another immune stimulant induced cytokine, cytokine receptor,cytokine receptor antagonist, chemokine, chemokine receptor, and/orchemokine receptor antagonist expression profile. In another embodiment,the one or more diagnostic biomarkers comprise inflammatory and/oranti-inflammatory cytokine. In other embodiments, the one or morediagnostic biomarkers comprise a cytokine receptor, cytokine receptorantagonist, chemokine, chemokine receptor, and/or chemokine receptorantagonist. In another embodiment, the diagnostic biomarkers areanalyzed in the presence of cortisol. In another embodiment, thecortisol concentration is in the range of 1 ug/ml to 500 ug/ml, with apreferred range of 1 ug/ml to 150 ug/ml. In another embodiment, the oneor more diagnostic biomarkers comprise a low expression of IL-10, IL-13and/or IL-1RA. In another embodiment, the low expression of IL-10, IL-13and/or IL-1RA comprises a 1 to 3 fold decrease in expression compared tolevels ordinarily found in a healthy individual.

There are many techniques readily available in the field for detectingthe presence or absence of a cytokine, cytokine receptor, cytokinereceptor antagonist, chemokine, chemokine receptor, and/or chemokinereceptor antagonist or other biomarkers, including protein microarrays.For example, some of the detection paradigms that can be employed tothis end include optical methods, electrochemical methods (voltometryand amperometry techniques), atomic force microscopy, and radiofrequency methods, e.g., magnetic resonance spectroscopy. Illustrativeof optical methods, in addition to microscopy, both confocal andnon-confocal, are detection of fluorescence, luminescence,chemiluminescence, absorbance, reflectance, transmittance, andbirefringence or refractive index (e.g., surface plasmon resonance,ellipsometry, a resonant mirror method, a grating coupler waveguidemethod or interferometry) such as ELISPOT.

Similarly, there are any numbers of techniques that may be employed toisolate and/or fractionate biomarkers. For example, a cytokine, cytokinereceptor, cytokine receptor antagonist, chemokine, chemokine receptor,and/or chemokine receptor antagonist may be captured using biospecificcapture reagents, such as antibodies, aptamers or antibodies thatrecognize the biomarker and modified forms of it. This method could alsoresult in the capture of protein interactors that are bound to theproteins or that are otherwise recognized by antibodies and that,themselves, can be biomarkers. The biospecific capture reagents may alsobe bound to a solid phase. Then, the captured proteins can be detectedby SELDI mass spectrometry or by eluting the proteins from the capturereagent and detecting the eluted proteins by traditional MALDI or bySELDI. One example of SELDI is called “affinity capture massspectrometry,” or “Surface-Enhanced Affinity Capture” or “SEAC,” whichinvolves the use of probes that have a material on the probe surfacethat captures analytes through a non-covalent affinity interaction(adsorption) between the material and the analyte. Some examples of massspectrometers are time-of-flight, magnetic sector, quadrupole filter,ion trap, ion cyclotron resonance, electrostatic sector analyzer andhybrids of these.

Alternatively, for example, the presence of biomarkers such as acytokine, cytokine receptor, cytokine receptor antagonist, chemokine,chemokine receptor, and/or chemokine receptor antagonist may be detectedusing traditional immunoassay techniques. Immunoassay requiresbiospecific capture reagents, such as antibodies, to capture theanalytes. The assay may also be designed to specifically distinguishprotein and modified forms of protein, which can be done by employing asandwich assay in which one antibody captures more than one form andsecond, distinctly labeled antibodies, specifically bind, and providedistinct detection of, the various forms. Antibodies can be produced byimmunizing animals with the biomolecules. Traditional immunoassays mayalso include sandwich immunoassays including ELISA or fluorescence-basedimmunoassays, as well as other enzyme immunoassays.

Prior to detection, a cytokine, cytokine receptor, cytokine receptorantagonist, chemokine, chemokine receptor, and/or chemokine receptorantagonist may also be fractionated to isolate them from othercomponents in a solution or of blood that may interfere with detection.Fractionation may include white blood cell isolation from other bloodcomponents, sub-cellular fractionation of white blood cell componentsand/or fractionation of the desired cytokine, cytokine receptor,cytokine receptor antagonist, chemokine, chemokine receptor, and/orchemokine receptor antagonist from other biomolecules found in whiteblood cells using techniques such as chromatography, affinitypurification, 1D and 2D mapping, and other methodologies forpurification known to those of skill in the art. In one embodiment, asample is analyzed by means of a biochip. Biochips generally comprisesolid substrates and have a generally planar surface, to which a capturereagent (also called an adsorbent or affinity reagent) is attached.Frequently, the surface of a biochip comprises a plurality ofaddressable locations, each of which has the capture reagent boundthere.

Alternatively, for example, the presence of biomarkers such as acytokine, cytokine receptor, cytokine receptor antagonist, chemokine,chemokine receptor, and/or chemokine receptor antagonist may be detectedusing PCR techniques or flow cytometry. All of the available techniquesfor cytokine, cytokine receptor, cytokine receptor antagonist,chemokine, chemokine receptor, and/or chemokine receptor antagonistdetection may be used.

Diagnostic Assays of the Invention

The invention provides various diagnostic assays/tests to predictwhether women are at risk of preterm birth.

As described above, in one embodiment of the invention, the diagnostictest comprises drawing blood from a patient, separating the blood cells(for example, separating the peripheral blood mononuclear cells (PBMC)),exposing the separated cells to an antigen (for example, LPS) andassessing the immune response.

In another embodiment, whole blood may be stimulated ex vivo withoutseparating the blood cells. For example, whole blood stimulation may becarried out by drawing blood from the subject into collection tubescoated with an antigen and/or stimulant and allowing it to incubate at37 degrees Celsius, for a desired amount of time (for example, for 16-24hours). The immune response may be subsequently assessed by usingvarious techniques known in the art. In one embodiment, after collectingblood from the subject and incubating with it with the antigen and/orstimulant, the collection tubes are centrifuged, the plasma is harvestedand assayed for an immune response.

In an embodiment, blood from the subject is drawn into collection tubescoated with antigens as well as into positive and negative controlcollection tubes. The antigens and/or stimulants induce an immuneresponse, for example by inducing expression of a cytokine, cytokinereceptor, cytokine receptor antagonist, chemokine, chemokine receptor,and/or chemokine receptor antagonist. The antigens or stimulants whichinduce the expression of a cytokine, cytokine receptor, cytokinereceptor antagonist, chemokine, chemokine receptor, and/or chemokinereceptor antagonist include but are not limited to LPS, cortisol,phytohemagglutinin, maternal histocompatibility antigens, paternalhistocompatibility antigens, fetal histocompatibility antigens, othermicrobial antigens, cytokines, phorbol esters (e.g., phorbol myristateacetate (PMA)), agents that induce Th1 or Th2 type differentiation oflymphocytes singly or combination thereof.

In a further embodiment, the immune response induced by the antigensand/or stimulants may be assessed by assaying the expression of acytokine, cytokine receptor, cytokine receptor antagonist, chemokine,chemokine receptor, and/or chemokine receptor antagonist. Varioustechniques may be used to assess the immune response including but notlimited to enzyme-linked immunosorbent spot (ELISpot), enzyme-linkedimmunosorbent assay (ELISA), microarray assay, quantitative PCR,Northern blot assay, Southern blot assay, Western blot assayimmunohistochemical assay or binding assay. In a preferred embodiment,ELISpot is used to assess the cytokine expression and/or chemokineexpression. These assays may be performed at bedsite/doctors' offices,in a laboratory or during transportation to the laboratory. Antibodiesagainst various cytokine, cytokine receptor, cytokine receptorantagonist, chemokine, chemokine receptor, and/or chemokine receptorantagonist may be used to assess an immune response. These antibodiesmay be any one or more of a monoclonal antibody or fragment thereof, apolyclonal antibody or a fragment thereof, chimeric antibodies,humanized antibodies, human antibodies and a single chain antibody. Inone embodiment, the antibodies may be conjugated with a radiolabel, anenzyme, a chromophore or a fluorophore for immune response detection.

To ascertain whether a woman is at an increased risk of pretermdelivery, the aforementioned assays are carried out using blood samplesfrom test subjects who are non-pregnant women and/or pregnant women. Theresults obtained (for example, the immune response induced which ismeasured as a function of the expression of a cytokine, cytokinereceptor, cytokine receptor antagonist, chemokine, chemokine receptor,and/or chemokine receptor antagonist) from the test subjects arecompared to the results obtained from control subjects. In oneembodiment, the control subjects are women who delivered at full term.In another embodiment, if the expression level of a cytokine, cytokinereceptor, cytokine receptor antagonist, chemokine, chemokine receptor,and/or chemokine receptor antagonist in test subjects is higher or lowerthan the range of the expression level of the cytokine, cytokinereceptor, cytokine receptor antagonist, chemokine, chemokine receptor,and/or chemokine receptor antagonist in the control subjects, the testsubject is at an increased risk of preterm delivery.

The invention further provides a diagnostic kit comprising a collectiontube coated with an antigen and/or stimulant, a positive controlcollection tube and/or a negative control collection tube. The kitfurther comprises reagents to detect an immune response (for exampleantibodies that bind to a cytokine, cytokine receptor, cytokine receptorantagonist, chemokine, chemokine receptor, and/or chemokine receptorantagonist expressed), for example, antibodies to detect the cytokine,cytokine receptor, cytokine receptor antagonist, chemokine, chemokinereceptor, and/or chemokine receptor antagonist, buffers andinstructions.

In various embodiments, the invention includes methods of identifying anon-pregnant woman at risk for preterm delivery, comprising: obtaining asample from the non-pregnant woman, determining the expression level ofone or more biomarkers in the sample, and comparing the expression levelof the one or more biomarkers with the expression level of biomarkersfrom those observed in women who delivered fullterm (controls). Sincethere is individual variation, the control values will constitute arange. If the level of expression of the one or more biomarkers in thesample is higher or lower than the level of expression of the samebiomarkers in women who delivered fullterm (outside the range), then itis indicative that the non-pregnant woman is at risk for pretermdelivery. Depending on how much the patient biomarker values are outsidethe range of the standard controls, the risk of preterm delivery may bedetermined as “low, medium or high.” The biomarkers include but are notlimited to IL-10, IL-13 and/or IL-1RA. The sample can comprisestimulated PBMC/whole blood supernatant (in the presence or absence ofcortisol). Alternatively, the sample can comprise serum.

In various embodiments, the invention includes methods of diagnosingsusceptibility of preterm delivery in a woman, comprising: obtaining asample from the woman, determining the expression level of one or morediagnostic biomarkers in the sample, and comparing the expression levelof the one or more biomarkers with the expression level of biomarkersfrom women who delivered fullterm (normal range). If the level ofexpression of the one or more diagnostic biomarkers in the sample ishigher or lower than the level of expression of the same diagnosticbiomarkers in the women who delivered fullterm (normal range), then itis indicative that the woman is at risk for preterm delivery. Thediagnostic biomarkers can be inflammatory and/or anti-inflammatorycytokines. In other embodiments, the diagnostic biomarkers can be acytokine receptor, cytokine receptor antagonist, chemokine receptor,and/or chemokine receptor antagonist. The diagnostic biomarkers selectedinclude but not limited to IL-10, IL-13 and/or IL-1RA. Biomarkerexpression levels that are at least one to three times less than (orgreater than, for other biomarkers) the biomarker expression levels of acontrol are indicative of preterm delivery. The sample can comprisestimulated PBMC/whole blood supernatant (in the presence or absence ofcortisol). Alternatively, the sample can comprise serum. The women maybe non-pregnant or pregnant.

The present invention is also directed to a kit for use to determine therisks of preterm delivery. The kit is useful for practicing theinventive method of determining the risk of preterm delivery ofnon-pregnant or pregnant women. The kit is an assemblage of materials orcomponents, including a diagnostic bioassay of the present invention.

The exact nature of the components configured in the inventive kitdepends on its intended purpose. For example, some embodiments areconfigured for the purpose of diagnosing susceptibility of women forpreterm delivery. In one embodiment, the kit is configured particularlyfor the purpose of determining the risk of preterm delivery ofnon-pregnant women. In another embodiment, the kit is configuredparticularly for the purpose of diagnosing susceptibility of women forpreterm delivery of pregnant or non-pregnant women.

Instructions for use may be included in the kit. “Instructions for use”typically include a tangible expression describing the technique to beemployed in using the components of the kit to effect a desired outcome,such as determine the risks of preterm delivery. Optionally, the kitalso contains other useful components, such as, specially prepared testtubes, diluents, buffers, pharmaceutically acceptable carriers,syringes, catheters, applicators, pipetting or measuring tools,bandaging materials or other useful paraphernalia as will be readilyrecognized by those of skill in the art.

The materials or components assembled in the kit can be provided to thepractitioner stored in any convenient and suitable ways that preservetheir operability and utility. For example the components can be indissolved, dehydrated, or lyophilized form; they can be provided atroom, refrigerated or frozen temperatures. The components are typicallycontained in suitable packaging material(s). As employed herein, thephrase “packaging material” refers to one or more physical structuresused to house the contents of the kit, such as inventive compositionsand the like. The packaging material is constructed by well-knownmethods, preferably to provide a sterile, contaminant-free environment.As used herein, the term “package” refers to a suitable solid matrix ormaterial such as glass, plastic, paper, foil, and the like, capable ofholding the individual kit components. Thus, for example, a package canbe a collection tube coated with an antigen and/or a stimulant for useto collect and stimulate a biological sample from a woman. The packagingmaterial generally has an external label which indicates the contentsand/or purpose of the kit and/or its components.

EXAMPLES

The following examples are provided to better illustrate the claimedinvention and are not to be interpreted as limiting the scope of theinvention. To the extent that specific materials are mentioned, it ismerely for purposes of illustration and is not intended to limit theinvention. One skilled in the art may develop equivalent means orreactants without the exercise of inventive capacity and withoutdeparting from the scope of the invention.

Example 1 Biomarker Assay

Peripheral blood mononuclear cells (PBMC) from non-pregnant (at least5-6 years post-partum) women with history of preterm or full termdelivery, in addition to the microbial component lipopolysaccharide(LPS)-induced cytokine expression profile were examined. PBMC wereseparated from whole blood using Ficoll gradient. The cells were countedand equal numbers of cells were plated in 24 well plates. PBMC weretreated with cortisol (50 or 300 ug/ml) or media for 1 hour prior tostimulation with LPS (0, 1, or 100 ug/ml) for 24 hours. The PBMC werelysed and the supernatant was examined for inflammatory andanti-inflammatory cytokine expression by using Bioplex technology(Bio-rad). The inventors found that during the non-pregnant state IL10,IL13 and IL1Ra expression was lower in the PBMC obtained from women whomhad previous preterm delivery, and that those biomarkers may be measuredto identify women who were at risk for preterm delivery in the future.

In this study two patients were preterm and four were full term. Therewere 9 samples per patient (3×3 design) and all samples from eachpatient were analyzed twice for cytokine concentrations with theexception of one full term subject who only had one analysis of eachsample. As a result there are a total of 99 observations for eachcytokine measured. The assay simultaneously measured concentrations of11 inflammatory markers: IL-1ra (IL1-receptor antagonist), IL-2, IL-4,IL-5, IL-8, IL-10, IL-12, IL-13, GM-CSF, IFN-g, and TNF-a.

Example 2 Establishment of Cut Off Points

Many samples were above or below the detection for some of theendpoints. Cut off points were established for each cytokine measured(Table 1). If a sample was out of the range of detection, a defaultvalue was assigned to the sample as indicated below.

TABLE 1 The establishment of cut off points for out of range values. Outof Fraction of Range Samples out of Cytokine Limit of Detection SetValue Detection Range IL-1ra all samples within levels n/a  0/99 ofdetection IL-2 LL = 0.50 0.25 10/99 IL-4 LL = 0.14 0.08  6/99 IL-5 LL =0.90 0.5 82/99 IL-8 UL = 95,000 100,000 79/99 IL-10 all samples withinlevels n/a  0/99 of detection IL-12 LL = 0.33 0.15 19/99 IL-13 LL = 0.170.08  8/99 GM-CSF LL = 1.18 0.60 11/99 IFN-g LL = 1.21 0.60  5/99 TNF-aall samples within levels n/a  0/99 of detection LL = Lower Limit; UL =Upper Limit

All samples that were below the range of detection for IL-2, IL-4,IL-13, and IFN-g were from the two subjects with a history of pretermdelivery. Of the 11 samples assayed that were below detection limits forGM-CSF, only 1 was from a subject with a full-term delivery while therest were from subjects with histories of preterm deliveries.

Example 3 Statistical Analysis

Empirical means and standard errors for each cytokine and treatmentgroup are presented herein. Here, the averages of replicate measureswere calculated first for each subject. The mean and SEM were then takenacross each treatment group.

All raw data was tested via the Kolmogorov-Smirnov test to determine ifthe data followed a normal distribution. Log-transformations wereperformed for all data found to have a non-normal distribution prior tofurther statistical analysis. For each cytokine, a mixed effects modelwas used to examine all data for significant effects with the outcomevariable as the cytokine concentration; the fixed predictor variables asLPS concentration, cortisol concentration, and delivery status (pre- orfull-term); and the random effect due to the replicate data points. Totest the interaction of the three fixed predictor variables on cytokineconcentration, both first and second level interactions ware added toeach cytokine model. Post-hoc testing was performed using a Student'st-test.

Example 4 Cortisol and LPS Concentrations

All 99 observations were initially used in the analysis to firstdetermine which concentrations of LPS and cortisol induced the mostrobust effect. For all cytokines measured there was no significantdifference between the effects of 1 ug/ml and 100 ug/ml LPS. For severalcytokines (IL-1ra, IL-10, and IL-13), 300 ug/ml cortisol had a moresignificant effect to suppress cytokine levels than 50 ug/ml.

As a result of the findings, the inventors focused on the data with 0 or300 ug/ml of cortisol pretreatment crossed with 0 or 1 ug/ml LPS. Thisresults in 4 samples per subject, each analyzed twice (with exception ofthe samples from the full term subject who only had one analysis of eachsample). With 8 measurements per subject (with the exception of the fullterm subject who only has 4 measurements) this results in a data setwith 44 measurements for each of the cytokines.

Example 5 Analysis of Testing for Cytokine Differences Between Groups

Using the same mixed model regression, estimated group means andstandard errors were calculated. Differences between preterm andfull-term pregnancies were considered significant where p<0.10.

Based on the results of the statistical modeling, cortisol did suppresssecretion of IL-13 and IL-1ra; LPS increased secretion of IL-4, IL-10,IL-13, TNF-a, and IL-1ra; and women who had preterm deliveries hadoverall lower secretion levels of IL-10, IL-13, and IL-1ra (Table 2).

TABLE 2 Global effects of cortisol, LPS, and premature delivery oncytokine secretion levels in PBMC based on results of mixed modeling.Cytokine Cortisol Effect LPS Effect Pre-term Effect IL-2 n/c, p = .80n/c, p = .50 n/c, p = .17 IL-4 n/c, p = .26 increase, p = .07 n/c, p =.59 IL-10 n/c, p = .12 increase, p = .01 decrease, p = .01 IL-13decrease, p = .01 increase, p = .04 decrease, p = .02 IFN-g n/c, p = .10n/c, p = .17 n/c, p = .22 TNF-a n/c, p = .41 increase, p = .04 n/c, p =.75 GM-CSF n/c, p = .51 n/c, p = .19 n/c, p = .17 IL-1ra decrease, p =.03 increase, p = .09 decrease, p = .01 n/c = No Change

Women who had preterm deliveries had lower baseline IL-10, 11,13 andIL-1Ra expression compared to those who delivered full term. There wereno differences in cytokine production between LPS-stimulated PBMC frompreterm delivering women when compared with that from women withfull-term deliveries. In other words, adjusting for unstimulatedsecretion levels, the concentration of cytokines released from LPSstimulation was no different between the two groups of women. In thepresence of cortisol PBMC from women who had preterm deliveries producedlower IL-13 and IFN-g, expression when compared with that from womenwith full-term deliveries.

REFERENCES

Hutzal C E, Boyle E M, Kenyon S L, Nash J V, Winsor 5, Taylor D J,Kirpalani H. Use of antibiotics for the treatment of preterm parturitionand prevention of neonatal morbidity: a metaanalysis. Am J ObstetGynecol. 2008 December; 199(6):620.e1-8. Epub 2008 Oct. 30.

Swamy G K, Ostbye T, Skjaerven R. Association of preterm birth withlong-term survival, reproduction, and next-generation preterm birth.JAMA. 2008 Mar. 26; 299(12):1429-36. Erratum in: JAMA. 2008 Jul. 9;300(2):170-1.

DeFranco E, Teramo K, Muglia L. Genetic influences on preterm birth.Semin Reprod Med. 2007 January; 25(1):40-51.

Plunkett J, Muglia L J. Genetic contributions to preterm birth:implications from epidemiological and genetic association studies. AnnMed. 2008; 40(3):167-95.

Goldenberg R L, Culhane J F, lams J D, Romero R. Epidemiology and causesof preterm birth. Lancet. 2008 Jan. 5; 371(9606):75-84.

Blank V, Hirsch E, Challis J R, Romero R, Lye S J. Cytokine signaling,inflammation, innate immunity and preterm labour—a workshop report.Placenta. 2008 March; 29 Suppl A:S102-4.

Mazaki-Tovi S, Romero R, Kusanovic J P, Erez O, Pineles B L, Gotsch F,Mittal P, Than N G, Espinoza J, Hassan S S. Recurrent preterm birth.Semin Perinatol. 2007 June; 31(3):142-58.

Romero R, Espinoza J, Gonçalves L F, Kusanovic J P, Friel L, Hassan S.The role of inflammation and infection in preterm birth. Semin ReprodMed, 2007 January; 25(1):21-39.

Romero R, Espinoza J, Gonçalves L F, Kusanovic J P, Friel L A, Nien J K,Inflammation in preterm and term labour and delivery. Semin FetalNeonatal Med. 2006 October; 11(5):317-26.

Luu T M, Ment L R, Schneider K C, Katz K H, Allan W C, Vohr B R. Lastingeffects of preterm birth and neonatal brain hemorrhage at 12 years ofage. Pediatrics. 2009 March; 123(3): 1037-44.

Morse S B, Zheng H, Tang Y, Roth J. Early school-age outcomes of latepreterm infants. Pediatrics. 2009 April; 123(4):e622-9.

Kamholz K L, Cole C H, Gray J E, Zupancic J A. Cost-effectiveness ofearly treatment for retinopathy of prematurity. Pediatrics. 2009January; 123(1):262-9.

Limperopoulos C, Bassan H, Sullivan N R, Soul J S, Robertson R L Jr,Moore M, Ringer S A, Volpe J J, du Plessis A J. Positive screening forautism in ex-preterm infants: prevalence and risk factors. Pediatrics,2008 April; 121(4):758-65.

Jacobs S E, O'Brien K, Inwood S, Kelly E N, Whyte H E. Outcome ofinfants 23-26 weeks of gestation pre and post surfactant. Acta Paediatr2000, 89(8):959-965.

Hack M, Flannery D J, Schluchter M, et al. Outcomes in young adulthoodfor very-low-birth-weight infants. N Engl J Med 2002, 346(3):149-157.

Genç, M R, Gerber S, Nesin M, Witkin S S. Polymorphism in theinterleukin-1 gene complex and spontaneous preterm delivery. Am J ObstetGynecol. 2002 July; 187(1):157-63.

Peltier M R. Immunology of term and preterm labor. Reprod BiolEndocrinol. 2003 Dec. 2; 1:122.

Kalish R B, Vardhana S, Gupta M, Perni S C, Witkin S S, Interleukin-4and -10 gene polymorphisms and spontaneous preterm birth in multifetalgestations. Am J Obstet Gynecol. 2004 March; 190(3):702-6.

Gotsch F, Romero R, Kusanovic J P, Erez O, Espinoza J, Kim C J, VaisbuchE, Than N G, Mazaki-Tovi S, Chaiworapongsa T, Mazor M, Yoon B H, EdwinS, Gomez R, Mittal P, Hassan S S, Sharma S. The anti-inflammatory limbof the immune response in preterm labor, intra-amnioticinfection/inflammation, and spontaneous parturition at term: a role forinterleukin-10. J Matern Fetal Neonatal Med. 2008 August; 21(8):529-47.

Berghella V, Hayes E, Visintine J, Baxter J K. Fetal fibronectin testingfor reducing the risk of preterm birth. Cochrane Database Syst Rev. 2008Oct. 8; (4):CD006843.

Herbst A, Nilsson C. Diagnosis of early preterm labour. BJOG. 2006December; 113 Suppl 3:60-7. Review. Erratum in: BJOG. 2008 April;115(5):674-5.

Lockwood C J, Senyei A E, Dische M R. Casal D, Shah K D, Thung S N,Jones L. Deligdisch L, Garite T J. Fetal fibronectin in cervical andvaginal secretions as a predictor of preterm delivery. N Engl J Med.1991 Sep. 5; 325(10):669-74.

Honest H, Bachmann L M, Gupta J K, Kleijnen J, Khan K S. Accuracy ofcervicovaginal fetal fibronectin test in predicting risk of spontaneouspreterm birth: systematic review. BMJ. 2002 Aug. 10; 325(7359):301.

Most O, Langer O, Kerner R, David G B, Calderon I. Can myometrialelectrical activity identify patients in preterm labor? Am J ObstetGynecol. 2008 October; 199(4):378.e1-6.

Menon R. Camargo M C, Thorsen P, Lombardi S J, Fortunato S J. Amnioticfluid interleukin-6 increase is an indicator of spontaneous pretermbirth in white but not black Americans. Am J Obstet Gynecol. 2008January; 198(1):77. e1-7.

Vogel I, Goepfert A R, Thorsen P, Skogstrand K, Hougaard D M, Curry A H,Cliver S, Andrews W W. Early second-trimester inflammatory markers andshort cervical length and the risk of recurrent preterm birth. J ReprodImmunol. 2007 October; 75(2):133-40. Epub 2007 Apr. 17.

Kim K W, Romero R, Park H S, Park C W, Shim S S, Jun J K, Yoon B H. Arapid matrix metalloproteinase-8 bedside test for the detection ofintraamniotic inflammation in women with preterm premature rupture ofmembranes. Am J Obstet Gynecol. 2007 September; 197(3):292.e1-5.

Spong C Y. Prediction and prevention of recurrent spontaneous pretermbirth. Obstet Gynecol. 2007 August; 110(2 Pt 1):405-15. Review.

Ghosh G. Breborowicz A, Brazert M, Maczkiewicz M, Kobelski M, Dubiel M,Gudmundsson S. Evaluation of third trimester uterine artery flowvelocity indices in relationship to perinatal complications. J MaternFetal Neonatal Med. 2006 September; 19(9):551-5.

Leitich H. Secondary predictors of preterm labour. BJOG. 2005 March; 112Suppl 1:48-50. Review.

Challis J R. Maternal corticotropin-releasing hormone, fetal growth, andpreterm birth. Am J Obstet Gynecol. 2004 October; 191(4):1059-60.

Heine R P, McGregor J A, Goodwin T M, Artal R, Hayashi R H, Robertson PA, Varner M W. Serial salivary estriol to detect an increased risk ofpreterm birth. Obstet Gynecol. 2000 October; 96(4):490.

Various embodiments of the invention are described above in the DetailedDescription. While these descriptions directly describe the aboveembodiments, it is understood that those skilled in the art may conceivemodifications and/or variations to the specific embodiments shown anddescribed herein. Any such modifications or variations that fall withinthe purview of this description are intended to be included therein aswell. Unless specifically noted, it is the intention of the inventorsthat the words and phrases in the specification and claims be given theordinary and accustomed meanings to those of ordinary skill in theapplicable art(s).

The foregoing description of various embodiments of the invention knownto the applicant at this time of filing the application has beenpresented and is intended for the purposes of illustration anddescription. The present description is not intended to be exhaustivenor limit the invention to the precise form disclosed and manymodifications and variations are possible in the light of the aboveteachings. The embodiments described serve to explain the principles ofthe invention and its practical application and to enable others skilledin the art to utilize the invention in various embodiments and withvarious modifications as are suited to the particular use contemplated.Therefore, it is intended that the invention not be limited to theparticular embodiments disclosed for carrying out the invention.

While particular embodiments of the present invention have been shownand described, it will be obvious to those skilled in the art that,based upon the teachings herein, changes and modifications may be madewithout departing from this invention and its broader aspects and,therefore, the appended claims are to encompass within their scope allsuch changes and modifications as are within the true spirit and scopeof this invention. It will be understood by those within the art that,in general, terms used herein are generally intended as “open” terms(e.g., the term “including” should be interpreted as “including but notlimited to,” the term “having” should be interpreted as “having atleast,” the term “includes” should be interpreted as “includes but isnot limited to,” etc.).

What is claimed is:
 1. A method, comprising: obtaining a biologicalsample from a woman who desires an assessment of her risk for pretermdelivery; exposing the biological sample ex vivo to an antigen and/or astimulant; and assessing an immune response to predict whether the womanis at risk for preterm delivery.
 2. The method of claim 1, furthercomprising separating blood cells from the sample and exposing theseparated blood cells to the antigen.
 3. The method of claim 1, whereinthe biological sample is whole blood.
 4. The method of claim 1, whereinexposing the biological sample ex vivo to the antigen or the stimulantis by collecting the biological sample into a container coated with theantigen and/or stimulant.
 5. The method of claim 4, further comprisingremoving plasma from the biological sample and assaying the plasma toassess the immune response.
 6. The method of claim 5, wherein removingplasma from the biological sample comprises centrifuging the tubecomprising the biological sample and harvesting the plasma.
 7. Themethod of claim 1, wherein the antigen and/or stimulant is cortisol,LPS, phytohemagglutinin, a maternal histocompatibility antigen, apaternal histocompatibility antigen, a fetal histocompatibility antigen,a microbial antigen, a cytokine, a phorbol ester, an agent capable ofinducing Th1 or Th2 type differentiation of lymphocytes or combinationthereof.
 8. The method of claim 1, wherein the antigen is LPS.
 9. Themethod of claim 1, wherein assessing the immune response comprisesassaying an expression level of a cytokine, cytokine receptor, cytokinereceptor antagonist, chemokine, chemokine receptor, chemokine receptorantagonist, or combinations thereof.
 10. The method of claim 9, whereinthe cytokine, cytokine receptor, cytokine receptor antagonist,chemokine, chemokine receptor, chemokine receptor antagonist is selectedfrom the group consisting of IL-10, IL-13, IL-1RA and combinationsthereof.
 11. The method of claim 9, wherein a higher or lower expressionlevel of the cytokine, cytokine receptor, cytokine receptor antagonist,chemokine, chemokine receptor, and/or chemokine receptor antagonistcompared to a control indicates that the woman is at an increased riskof preterm delivery.
 12. The method of claim 11, wherein the higher orlower expression level of the cytokine, cytokine receptor, cytokinereceptor antagonist, chemokine, chemokine receptor, and/or chemokinereceptor antagonist is at least one to three times higher or lowercompared to the levels of the control.
 13. The method of claim 11,wherein the level of the higher or lower expression of the cytokine,cytokine receptor, cytokine receptor antagonist, chemokine, chemokinereceptor, and/or chemokine receptor antagonist is indicative of a low,medium, or high risk of preterm delivery.
 14. The method of claim 9,wherein a statistically significantly higher or lower expression of thecytokine, cytokine receptor, cytokine receptor antagonist, chemokine,chemokine receptor, and/or chemokine receptor antagonist compared to acontrol indicates that the woman is at an increased risk of pretermdelivery.
 15. The method of claim 1, wherein the assessing the immuneresponse comprising using a technique selected from the group consistingof enzyme-linked immunosorbent spot (ELISpot), enzyme-linkedimmunosorbent assay (ELISA), microarray assay, quantitative BCP,Northern blot assay, Southern blot assay, Western blot assay,immunohistochemical assay, binding assay and combinations thereof.
 16. Akit, comprising: a container comprising an antigen and/or a stimulant;instructions to use the container to collect a biological sample from awoman to detect an immune response; and instructions to determine therisk of preterm delivery of the woman.
 17. The kit of claim 16, furthercomprising a reagent to detect the immune response.
 18. The kit of claim16, further comprising a positive control and/or a negative control. 19.A method, comprising: obtaining a sample of whole blood from a woman whodesires an assessment of her risk for preterm delivery; exposing thesample ex vivo to an antigen and/or a stimulant by collecting the sampleinto a container coated with the antigen and/or stimulant; and assessingan immune response to predict whether the woman is at risk for pretermdelivery.
 20. The method of claim 19, wherein the antigen and/orstimulant is cortisol, LPS, phytohemagglutinin, or combinations thereof.21. The method of claim 19, wherein assessing the immune responsecomprises assaying an expression level of a cytokine, cytokine receptor,cytokine receptor antagonist, chemokine, chemokine receptor, chemokinereceptor antagonist, or combinations thereof, wherein a statisticallysignificantly higher or lower expression level of the cytokine, cytokinereceptor, cytokine receptor antagonist, chemokine, chemokine receptor,and/or chemokine receptor antagonist compared to a control indicatesthat the woman is at an increased risk of preterm delivery.
 22. Themethod of claim 21, wherein the cytokine, cytokine receptor, cytokinereceptor antagonist, chemokine, chemokine receptor, chemokine receptorantagonist is selected from the group consisting of IL-10, IL-13, IL-1RAand combinations thereof.